This application claims priority of German Application No. 101 08 796.9, filed Feb. 21, 2001, the complete disclosure of which is hereby incorporated by reference.
The aperture is an essential criterion for characterizing the performance of microscope objectives. It is the product of the sine of half the acceptance angle and the refractive index of the immersion liquid in immersion applications. The acceptance angle is significant particularly in TIRF (Total Internal Reflection Fluorescence) applications, where total reflection is used to observe fluorescence in an interface or boundary layer. In fluorescence excitation by total reflection (TIRF), the objective is used in an aqueous specimen medium. Collimated light is coupled in at one point in the pupil of the objective which corresponds to an aperture of  greater than 1.38. This aperture exceeds the critical angle of total reflection of the coverslip/water transition, i.e., the incident light is reflected at the boundary surface by total reflection; as a result, there occurs directly above the boundary layer an evanescent light wave, as it is called, whose penetration depth in water is only on the order of magnitude of the light wavelength. This enables depth-selective fluorescence excitation of objects in the immediate vicinity of the boundary layer.
Prior patent publications on microscope objectives with standard immersions do not substantially exceed apertures of more than 1.4. The higher-aperture systems ( greater than 1.4) described in the patent literature achieve this via the refractive index of the immersion liquid (n approximately 1.78) (U.S. Pat. No. 5,659,425). However, immersion liquids of the type mentioned above have considerable disadvantages, so that they are applied only seldom in practice (toxic, inhomogeneous, etc.). In addition, the refractive index of the coverslip material must also be adapted, which entails additional cost.
It is the primary object of the invention to achieve an objective with a high aperture and simple construction.
The present invention achieves a surprisingly large acceptance angle in a simple construction, i.e., compared with other microscope objectives, the quotient of the numerical aperture and the index of refraction of the immersion oil (standard immersion oils approximately 1.5) is not exceeded.
With standard immersion oils (n[546 nm]=1.52), the aperture has a value in the axis of about 1.45 and still has a value of about 1.43 at the edge of field (2yxe2x80x2=20 mm). Substantial advantages such as better detail resolution and higher light intensity are achieved through the large aperture.